The Role of SOX2 and SOX9 Transcription Factors in the Reactivation-Related Functional Properties of NT2/D1-Derived Astrocytes.

Astrocytes are the main homeostatic cells in the central nervous system, with the unique ability to transform from quiescent into a reactive state in response to pathological conditions by reacquiring some precursor properties. This process is known as reactive astrogliosis, a compensatory response that mediates tissue damage and recovery. Although it is well known that SOX transcription factors drive the expression of phenotype-specific genetic programs during neurodevelopment, their roles in mature astrocytes have not been studied extensively. We focused on the transcription factors SOX2 and SOX9, shown to be re-expressed in reactive astrocytes, in order to study the reactivation-related functional properties of astrocytes mediated by those proteins. We performed an initial screening of SOX2 and SOX9 expression after sensorimotor cortex ablation injury in rats and conducted gain-of-function studies in vitro using astrocytes derived from the human NT2/D1 cell line. Our results revealed the direct involvement of SOX2 in the reacquisition of proliferation in mature NT2/D1-derived astrocytes, while SOX9 overexpression increased migratory potential and glutamate uptake in these cells. Our results imply that modulation of SOX gene expression may change the functional properties of astrocytes, which holds promise for the discovery of potential therapeutic targets in the development of novel strategies for tissue regeneration and recovery.

Interactions of ultrashort laser pulses with hemoglobin: Photophysical aspects and potential applications.

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

The Effect of Liquid-Phase Exfoliated Graphene Film on Neurodifferentiation of Stem Cells from Apical Papilla.

BACKGROUND: Dental stem cells, which originate from the neural crest, due to their easy accessibility might be good candidates in neuro-regenerative procedures, along with graphene-based nanomaterials shown to promote neurogenesis in vitro. We aimed to explore the potential of liquid-phase exfoliated graphene (LPEG) film to stimulate the neuro-differentiation of stem cells from apical papilla (SCAP). METHODS: The experimental procedure was structured as follows: (1) fabrication of graphene film; (2) isolation, cultivation and SCAP stemness characterization by flowcytometry, multilineage differentiation (osteo, chondro and adipo) and quantitative PCR (qPCR); (3) SCAP neuro-induction by cultivation on polyethylene terephthalate (PET) coated with graphene film; (4) evaluation of neural differentiation by means of several microscopy techniques (light, confocal, atomic force and scanning electron microscopy), followed by neural marker gene expression analysis using qPCR. RESULTS: SCAP demonstrated exceptional stemness, as judged by mesenchymal markers' expression (CD73, CD90 and CD105), and by multilineage differentiation capacity (osteo, chondro and adipo-differentiation). Neuro-induction of SCAP grown on PET coated with graphene film resulted in neuron-like cellular phenotype observed under different microscopes. This was corroborated by the high gene expression of all examined key neuronal markers (Ngn2, NF-M, Nestin, MAP2, MASH1). CONCLUSIONS: The ability of SCAPs to differentiate toward neural lineages was markedly enhanced by graphene film.

Tenascin-C fibronectin D domain is involved in the fine-tuning of glial response to CNS injury in vitro.

Understanding processes that occur after injuries to the central nervous system is essential in order to gain insight into how the restoration of function can be improved. Extracellular glycoprotein tenascin-C (TnC) has numerous functions in wound healing process depending on the expression time, location, isoform and binding partners which makes it interesting to study in this context. We used an in vitro injury model, the mixed culture of cortical astrocytes and microglia, and observed that without TnC microglial cells tend to populate gap area in greater numbers and proliferate more, whereas astrocytes build up in the border region to promote faster gap closure. Alternatively spliced domain of TnC, fibronectin type III-like repeat D (FnD) strongly affected physiological properties and morphology of both astrocytes and microglia in this injury model. The rate of microglial proliferation in the injury region decreased significantly with the addition of FnD. Additionally, density of microglia also decreased, in part due to reduced proliferation, and possibly due to reduced migration and increased contact inhibition between enlarged FnD-treated cells. Overall morphology of FnD-treated microglia resembled the activated pro-inflammatory cells, and elevated expression of iNOS was in accordance with this phenotype. The effect of FnD on astrocytes was different, as it did not affect their proliferation, but stimulated migration of reactivated astrocytes into the scratched area 48 h after the lesion. Elevated expression and secretion of TNF-α and IL-1ß upon FnD treatment indicated the onset of inflammation. Furthermore, on Western blots we observed increased intensity of precursor bands of ß1 integrin and appearance of monomeric bands of P2Y12R after FnD treatment which substantiates and clarifies its role in cellular shape and motility changes. Our results show versatile functions of TnC and in particular FnD after injury, mostly contributing to ongoing inflammation in the injury region. Based on our findings, FnD might be instrumental in limiting immune cell infiltration, and promoting astrocyte migration within the injury region, thus influencing spaciotemporal organization of the wound and surrounding area.

Reactive and Senescent Astroglial Phenotypes as Hallmarks of Brain Pathologies.

Astrocytes, as the most abundant glial cells in the central nervous system, are tightly integrated into neural networks and participate in numerous aspects of brain physiology and pathology. They are the main homeostatic cells in the central nervous system, and the loss of astrocyte physiological functions and/or gain of pro-inflammatory functions, due to their reactivation or cellular senescence, can have profound impacts on the surrounding microenvironment with pathological outcomes. Although the importance of astrocytes is generally recognized, and both senescence and reactive astrogliosis have been extensively reviewed independently, there are only a few comparative overviews of these complex processes. In this review, we summarize the latest data regarding astrocyte reactivation and senescence, and outline similarities and differences between these phenotypes from morphological, functional, and molecular points of view. A special focus has been given to neurodegenerative diseases, where these phenotypic alternations of astrocytes are significantly implicated. We also summarize current perspectives regarding new advances in model systems based on astrocytes as well as data pointing to these glial cells as potential therapeutic targets.

Vitamin B Complex and Experimental Autoimmune Encephalomyelitis -Attenuation of the Clinical Signs and Gut Microbiota Dysbiosis.

The present study aimed to investigate the neuroprotective effects of the vitamin B complex (B1, B2, B3, B5, B6, and B12-VBC), by studying the changes in the femoral nerve, quadriceps muscle, popliteal lymph nodes and gut microbiota in the rat model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). VBC treatment attenuated clinical signs of EAE during the disease, and reduced the duration of EAE thereby contributing to a faster recovery. In VBC-treated EAE rats, a significant decrease in nerve and muscle nuclear density was revealed during the onset period of the disease, while a marked increase was detected at the end of the disease, compared with untreated EAE rats. In the lymph nodes of VBC-treated EAE rats, a fewer number of lymphoid follicles in the cortical area and smaller epithelioid granulomas were detected. The changes in microbiota composition were examined using 16S rRNA gene sequencing and bioinformatics analysis, which revealed the potential of VBC treatment in establishing and/or maintaining gut microbiota homeostasis. Finally, the present study demonstrated that VBC treatment ameliorated the cellular changes in the affected peripheral nerve, muscles innervated by this nerve, and the gut microbiota dysbiosis which occurred during the EAE.

Approach for patch-clamping using an upright microscope with z-axis movable stage.

We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch-clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z-axis movable stage and a fixed objective can also be efficiently used in combination with the patch-clamp technique. We define a set of underlying principles governing the operation of this microscope/patch-clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole-cell patch-clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost-effective in reducing the investments in purchasing additional equipment. We provide step-by-step instructions on implementing an upright microscope with z-axis movable stage as a routine workhorse for patch-clamping. RESEARCH HIGHLIGHTS: Approach for efficient patch-clamping using an upright microscope with a z-axis movable stage. Routine study of live cell physiology. Custom microscope/patch-clamp configuration that is cost-effective and overcomes equipment limitations.

Cysteine Peptidase Cathepsin X as a Therapeutic Target for Simultaneous TLR3/4-mediated Microglia Activation.

Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.

Dysfunction of oligodendrocyte inwardly rectifying potassium channel in a rat model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the death of motor neurons in the spinal cord and the brain. Although this disease is characterized by motoneuron degeneration, non-neuronal cells such as oligodendrocytes play an important role in the disease onset and progression. The aim of our study was to examine functional properties of oligodendrocytes in the SOD1G93A rat model of ALS with a particular focus on the inwardly rectifying potassium channel Kir4.1 that is abundantly expressed in these glial cells and plays a role in the regulation of extracellular K+ . First, we demonstrate that the expression of Kir4.1 is diminished in the spinal cord oligodendrocytes of the SOD1G93A rat. Moreover, our data show an elevated number of dysmorphic oligodendrocytes in the ALS spinal cord that is indicative of a degenerative phenotype. In order to assess physiological properties of oligodendrocytes, we prepared cell cultures from the rat spinal cord. Oligodendrocytes isolated from the SOD1G93A spinal cord display similar ramification of the processes as the control but express a lower level of Kir4.1. We further demonstrate an impairment of oligodendrocyte functional properties in ALS. Remarkably, whole-cell patch-clamp recordings revealed compromised membrane biophysical properties and diminished inward currents in the SOD1G93A oligodendrocytes. In addition, the Ba2+ -sensitive Kir currents were decreased in ALS oligodendrocytes. Altogether, our findings provide the evidence of impaired Kir4.1 expression and function in oligodendrocytes of the SOD1G93A spinal cord, suggesting oligodendrocyte Kir4.1 channel as a potential contributor to the ALS pathophysiology.

Corrigendum: Different Functions of Recombinantly Expressed Domains of Tenascin-C in Glial Scar Formation.

[This corrects the article DOI: 10.3389/fimmu.2020.624612.].

The Electrophysiological Determinants of Corticospinal Motor Neuron Vulnerability in ALS.

The brain is complex and heterogeneous. Even though numerous independent studies indicate cortical hyperexcitability as a potential contributor to amyotrophic lateral sclerosis (ALS) pathology, the mechanisms that are responsible for upper motor neuron (UMN) vulnerability remain elusive. To reveal the electrophysiological determinants of corticospinal motor neuron (CSMN, a.k.a UMN in mice) vulnerability, we investigated the motor cortex of hSOD1G93A mice at P30 (postnatal day 30), a presymptomatic time point. Glutamate uncaging by laser scanning photostimulation (LSPS) revealed altered dynamics especially within the inhibitory circuitry and more specifically in L2/3 of the motor cortex, whereas the excitatory microcircuits were unchanged. Observed microcircuitry changes were specific to CSMN in the motor column. Electrophysiological evaluation of the intrinsic properties in response to the microcircuit changes, as well as the exon microarray expression profiles of CSMN isolated from hSOD1G93A and healthy mice at P30, revealed the presence of a very dynamic set of events, ultimately directed to establish, maintain and retain the balance at this early stage. Also, the expression profile of key voltage-gated potassium and sodium channel subunits as well as of the inhibitory GABA receptor subunits and modulatory proteins began to suggest the challenges CSMN face at this early age. Since neurodegeneration is initiated when neurons can no longer maintain balance, the complex cellular events that occur at this critical time point help reveal how CSMN try to cope with the challenges of disease manifestation. This information is critically important for the proper modulation of UMNs and for developing effective treatment strategies.

Different Functions of Recombinantly Expressed Domains of Tenascin-C in Glial Scar Formation.

Extracellular matrix glycoprotein tenascin-C (TnC) is highly expressed in vertebrates during embryonic development and thereafter transiently in tissue niches undergoing extensive remodeling during regeneration after injury. TnC's different functions can be attributed to its multimodular structure represented by distinct domains and alternatively spliced isoforms. Upon central nervous system injury, TnC is upregulated and secreted into the extracellular matrix mainly by astrocytes. The goal of the present study was to elucidate the role of different TnC domains in events that take place after spinal cord injury (SCI). Astrocyte cultures prepared from TnC-deficient (TnC-/-) and wild-type (TnC+/+) mice were scratched and treated with different recombinantly generated TnC fragments. Gap closure, cell proliferation and expression of GFAP and cytokines were determined in these cultures. Gap closure in vitro was found to be delayed by TnC fragments, an effect mainly mediated by decreasing proliferation of astrocytes. The most potent effects were observed with fragments FnD, FnA and their combination. TnC-/- astrocyte cultures exhibited higher GFAP protein and mRNA expression levels, regardless of the type of fragment used for treatment. Application of TnC fragments induced also pro-inflammatory cytokine production by astrocytes in vitro. In vivo, however, the addition of FnD or Fn(D+A) led to a difference between the two genotypes, with higher levels of GFAP expression in TnC+/+ mice. FnD treatment of injured TnC-/- mice increased the density of activated microglia/macrophages in the injury region, while overall cell proliferation in the injury site was not affected. We suggest that altogether these results may explain how the reaction of astrocytes is delayed while their localization is restricted to the border of the injury site to allow microglia/macrophages to form a lesion core during the first stages of glial scar formation, as mediated by TnC and, in particular, the alternatively spliced FnD domain.

The down-regulation of Notch 1 signaling contributes to the severity of bone loss in aggressive periodontitis.

BACKGROUND: The exact mechanisms of bone resorption in periodontitis have not been fully elucidated. The aims of this study were to analyze the expression of Notch signaling molecules, bone remodeling mediators, and pro-inflammatory cytokines in periodontitis patients and to determine their potential correlations. METHODS: The study included 130 individuals: 40 with aggressive periodontitis (AP group), 40 with chronic periodontitis (CP group), and 50 periodontally healthy controls. Total RNA was extracted from gingival crevicular fluid samples and relative gene expression of investigated molecules (Notch 1, Notch 2, Jagged 1, Hes 1, Hey 1, TNF-α, IL-17, RANKL, and OPG) was determined by reverse transcriptase - real-time polymerase chain reaction (RT-qPCR). RESULTS: In AP group, a significant increase of Notch 2, TNF-α, IL-17 and RANKL and a significant decrease of Notch 1 and Jagged 1 expression were observed compared to control group (P = 0.023, P = 0.005, P = 0.030, and P = 0.001 P = 0.031 and P = 0.029, respectively). Notch 2 and RANKL were also overexpressed in CP group compared to controls (P = 0.001 and P = 0.011). Significant correlations were observed in AP group between expression levels of the analyzed genes. CONCLUSION: The present findings implicate Notch 2 overexpression in the ethiopathogenesis of bone resorption in aggressive and chronic periodontitis. The down-regulation of Notch 1 and Jagged 1 and loss of their osteoprotective function might cause a more excessive osteoclast formation and contribute to greater osteolysis in aggressive periodontitis.

The S100A4 Transcriptional Inhibitor Niclosamide Reduces Pro-Inflammatory and Migratory Phenotypes of Microglia: Implications for Amyotrophic Lateral Sclerosis.

S100A4, belonging to a large multifunctional S100 protein family, is a Ca2+-binding protein with a significant role in stimulating the motility of cancer and immune cells, as well as in promoting pro-inflammatory properties in different cell types. In the CNS, there is limited information concerning S100A4 presence and function. In this study, we analyzed the expression of S100A4 and the effect of the S100A4 transcriptional inhibitor niclosamide in murine activated primary microglia. We found that S100A4 was strongly up-regulated in reactive microglia and that niclosamide prevented NADPH oxidase 2, mTOR (mammalian target of rapamycin), and NF-κB (nuclear factor-kappa B) increase, cytoskeletal rearrangements, migration, and phagocytosis. Furthermore, we found that S100A4 was significantly up-regulated in astrocytes and microglia in the spinal cord of a transgenic rat SOD1-G93A model of amyotrophic lateral sclerosis. Finally, we demonstrated the increased expression of S100A4 also in fibroblasts derived from amyotrophic lateral sclerosis (ALS) patients carrying SOD1 pathogenic variants. These results ascribe S100A4 as a marker of microglial reactivity, suggesting the contribution of S100A4-regulated pathways to neuroinflammation, and identify niclosamide as a possible drug in the control and attenuation of reactive phenotypes of microglia, thus opening the way to further investigation for a new application in neurodegenerative conditions.

Differentiation of stem cells from apical papilla into neural lineage using graphene dispersion and single walled carbon nanotubes.

Stem cell-based therapies are considered a promising treatment modality for many medical conditions. Several types of stem cells with variable differentiation potentials have been isolated from dental tissues, among them stem cells from apical papilla (SCAP). In parallel, new classes of biocompatible nanomaterials have also been developed, including graphene and carbon nanotube-based materials. The aim of the study was to assess whether graphene dispersion (GD) and water-soluble single walled carbon nanotubes (ws-SWCNT), may enhance SCAPs capacity to undergo neural differentiation. SCAPs cultivated in neuroinductive medium supplemented with GD and ws-SWCNT, separately and in combination, were subjected to neural marker analysis by real-time polymerase chain reaction (neurofilament medium [NF-M], neurogenin-2 [ngn-2], ß III-tubulin, microtubule-associated protein 2) and immunocytochemistry (NeuN and ß III-tubulin). GD, ws-SWCNT, and their combination, had neuro-stimulatory effects on SCAPs, as judged by the production of neural markers. Compared to cells grown in nanomaterial free medium, cells with GD showed higher production of B3T, cells with ws-SWCNT had higher production of ngn-2 and NF-M, while the combination of nanomaterials gave similar levels of both B3T and NF-M as the neuroinductive medium alone, but with the finest neuron-like morphology. In conclusion, GD and ws-SWCNT seem to enhance neural differentiation of SCAP. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2653-2661, 2018.

The Astrocytic S100B Protein with Its Receptor RAGE Is Aberrantly Expressed in SOD1G93A Models, and Its Inhibition Decreases the Expression of Proinflammatory Genes.

Neuroinflammation is one of the major players in amyotrophic lateral sclerosis (ALS) pathogenesis, and astrocytes are significantly involved in this process. The astrocytic protein S100B can be released in pathological states activating the receptor for advanced glycation end products (RAGE). Different indications point to an aberrant expression of S100B and RAGE in ALS. In this work, we observed that S100B and RAGE are progressively and selectively upregulated in astrocytes of diseased rats with a tissue-specific timing pattern, correlated to the level of neurodegeneration. The expression of the full-length and soluble RAGE isoforms could also be linked to the degree of tissue damage. The mere presence of mutant SOD1 is able to increase the intracellular levels and release S100B from astrocytes, suggesting the possibility that an increased astrocytic S100B expression might be an early occurring event in the disease. Finally, our findings indicate that the protein may exert a proinflammatory role in ALS, since its inhibition in astrocytes derived from SOD1G93A mice limits the expression of reactivity-linked/proinflammatory genes. Thus, our results propose the S100B-RAGE axis as an effective contributor to the pathogenesis of the disease, suggesting its blockade as a rational target for a therapeutic intervention in ALS.

Targeting Astrocytes for Treatment in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder affecting upper and lower motoneurons. The two types, sporadic and familial differ in the aetiopathogenesis but have a similar neuropathology characterized by oxidative stress, excitotoxicity and inflammation. The disease is also characterized by a non-cell autonomous mechanism with astrocytes playing a central role by affecting synaptic glutamate, the blood-brain barrier, and metabolic and trophic support. Two types of therapeutic approaches focusing on astrocytes are presented: a) emerging molecular targets (potassium inward rectifier channels and aquaporins at the astrocyte endfeet, and IP3 receptor signaling pathway), and b) cell therapy with stem cell - generated and transplanted astrocytes.

Short-term fasting promotes insulin expression in rat hypothalamus.

In the hypothalamus, insulin takes on many roles involved in energy homoeostasis. Therefore, the aim of this study was to examine hypothalamic insulin expression during the initial phase of the metabolic response to fasting. Hypothalamic insulin content was assessed by both radioimmunoassay and Western blot. The relative expression of insulin mRNA was examined by qPCR. Immunofluorescence and immunohistochemistry were used to determine the distribution of insulin immunopositivity in the hypothalamus. After 6-h fasting, both glucose and insulin levels were decreased in serum but not in the cerebrospinal fluid. Our study showed for the first time that, while the concentration of circulating glucose and insulin decreased, both insulin mRNA expression and insulin content in the hypothalamic parenchyma were increased after short-term fasting. Increased insulin immunopositivity was detected specifically in the neurons of the hypothalamic periventricular nucleus and in the ependymal cells of fasting animals. These novel findings point to the complexity of mechanisms regulating insulin expression in the CNS in general and in the hypothalamus in particular.